Yang_2025_Appl.Biochem.Biotechnol__

Methyl (R, S)-2, 6-dimethylphenylaminopropionate ((R, S)-1) is an intermediate in the production of the agricultural fungicide (R, S)-metalaxyl. (R, S)-1 can be hydrolyzed enantioselectively by some hydrolases to produce (R)-1, which was used for the production of (R)-metalaxyl. In this work, a strain Burkholderia sp. YD106 that could hydrolyze (R, S)-1 was screened from the activated sludge, but it had almost no enantioselectivity. The intracellular active esterase WZest was successfully heterologously expressed in the recombinant E. coli BL21 (DE3)-pET-28a ( +)-GE04845. Using the recombinant strain as the parent strain, the mutants were constructed by a site-directed mutation. Among all 33 mutants, seven had altered enantioselectivity, of which four mutants were (R)-enantioselective and three were (S)-enantioselective. The mutant WZest-W23T had the highest (R)-enantioselectivity. When it catalyzed the hydrolysis of (R, S)-1 at 44.6% substrate conversion, e.e.(p) reached 94.70% with an enantiomeric ratio (E) of 85.0. WZest showed significant amino acid sequence differences from the two reported esterases capable of hydrolyzing (R, S)-1. It was both active in two kinds of solutions. One was an emulsion with the substrate (R, S)-1 emulsified with Tween80, and the other was a homogeneous solution with acetone as a co-solvent. The activity of WZest in the former was higher than that in the latter.